Site-directed mutagenesis modified glycoprotein hormones and methods of use

ABSTRACT

Described are new glycoprotein hormones capable of competing with natural hormones for the normal receptor binding sites but substantially incapable of effecting post receptor activities. The glycoprotein hormones of the present invention have had specific (rather than all) oligosaccharide chains removed so as to effectively diminish biologic activity while not significantly reducing plasma half-life, thus improving the molecules effectiveness as an antagonist compared with conventionally-produced molecules. The preferred glycoprotein hormones are ideally obtained by site-directed mutagenesis to selectively deglycosylate the protein. Also described are therapeutic treatments comprising the administration of the recombinant glycoprotein hormones of the present invention as hormone antagonists.

The present application is a divisional of U.S. application Ser. No.08/126,134, filed Sep. 23, 1993, now U.S. Pat. No. 5,352,779, which is adivisional of U.S. application Ser. No. 07/136,236, filed Dec. 21, 1987,now U.S. Pat. No. 5,260,421.

FIELD OF THE INVENTION

This invention relates generally to hormones and more particularly toglycoprotein hormones including the pituitary glycoproteins, and furtherprovides methods for creating hormone analogs by site-directedmutagenesis.

BACKGROUND OF THE INVENTION

Glycoprotein hormones, especially those synthesized and secreted by theanterior pituitary gland, play critically important roles in a varietyof bodily functions including: metabolism, temperature regulation,growth, and reproduction. The pituitary glycoproteins, luteinizinghormone (LH), follicle stimulating hormone (FSH), and thyroidstimulating hormone (TSH) are similar in structure to the placentalgonadotropin, human chorionic gonadotropin (hCG). Each of the moleculesis actually a dimer consisting of two protein chains held together bynon-covalent, ionic interactions. The alpha chain for each of thehormones is identical. The beta chain is the hormone-specific portion ofthe dimer.

Following secretion, the hormones travel in the blood stream to thetarget cells which contain membrane bound receptors. The hormone bindsto the receptor and stimulates the cell. Typically, such stimulationinvolves an increase in activity of a specific intracellular regulatoryenzyme which in turn catalyzes a biochemical reaction essential to theresponse of the cell. In the case of hCG, binding to the hCG receptorpresent upon the corpus luteum (an ovarian structure), stimulates theactivity of the enzyme adenylate cyclase. This enzyme catalyzes theconversion of intracellular ATP to cyclic AMP (cAMP). cAMP stimulatesthe activity of other enzymes involved in the production of ovariansteroid hormones, especially progesterone. hCG-stimulated progesteronesecretion is essential for the maintenance of pregnancy during the firsttrimester of gestation.

The exact mechanism by which a dimeric glycoprotein hormone, such ashCG, stimulates post-receptor events, such as activation of adenylatecyclase activity, is unknown. By a variety of experimentalmanipulations, it has been shown, however, that the carbohydratestructures, each attached to the hCG molecule by a linkage at respectiveasparagine residues (N-linked), play important roles in this regard.Treatment of glycoprotein hormones, such as LH, FSH, or hCG withhydrogen fluoride removes approximately 70 percent of theoligosaccharide side chains. The resultant partially deglycosylatedmolecules retain their receptor binding activity but are unable tostimulate any post-receptor events. Thus it is clear that the sugarportion of the glycoprotein hormone, while not directly involved withreceptor binding, plays a critical role in post-receptor events, andtherefore, biologic activity.

It is also known that in addition to a role in the in vitro bioactivity,oligosaccharides are important components of the molecule's survivaltime in circulation. Indeed, plasma half-life of a glycoprotein hormoneis directly related to the amount of one particular sugar molecule,sialic acid, generally present upon the most distal portion of theoligosaccharide chain. The removal of the carbohydrate portions (byhydrogen fluoride treatment) would result in the production of hormonesthat bind to the receptor but fail to exert the expected biologicresponse. In addition, these molecules would have an extremely shortplasma half-life since the lack of terminal sialic acid residues wouldincrease the binding affinity to the hepatic asialoglycoprotein receptorthereby hastening clearance from the systemic circulation.

Many clinical endocrinopathies are the result of over production ofstimulating hormones (e.g., excess TSH secretion resulting inhyperthyroidism). A conventional treatment for a pathologic state causedby a hormone excess would be the administration of a hormone antagonist.To be effective, an antagonist must bind with high affinity to thereceptor but fail to activate post receptor events. From the earlierdiscussion, it can be anticipated that hydrogen fluoride treatedhormones (that have had approximately 70 percent of carbohydratesremoved) would be effective, competitive antagonists. Indeed, it hasbeen shown that HF-treated hormones bind with somewhat greater affinityfor the biologic receptor, compete effectively with native material, anddiminish the expected biologic action of native hormone in adose-dependent fashion.

At first glance, such a hormone preparation would appear to be a viablecandidate for a competitive antagonist therapeutic agent. However, fourproblems are associated with large scale production of suchpreparations. First, all pituitary hormone preparations are generallycontaminated with other hormones. Thus, while it may be possible toobtain a preparation of a hormone and partially deglycosylate it; theresultant preparation would also contain other deglycosylated hormonesas contaminants which may disadvantageously produce unwanted andunacceptable side effects following administration. Secondly,preparations of partially purified hormones vary greatly in theirpotency, physicochemical characteristics and purity. Therefore, eachbatch produced would need to be analyzed carefully. The possibility ofbatch-to-batch variability would necessitate repeated execution ofclinical trials to determine the effective dose. Thirdly, the methodused to deglycosylate is incomplete and, therefore, somewhatuncontrollable. No information is available as to the variabilityassociated with hydrogen fluoride treatment of successive batches ofhormone. Potential variability in this process would also requireextensive characterization of each batch produced. Fourth, carbohydrateside chains also play an important role in dictating the plasmahalf-life of a molecule. Partially deglycosylated hormones have beenshown to be rapidly cleared from the circulation following injection.Therefore, repeated injections of large doses of deglycosylated hormoneswould be required to achieve a desired effect. The need for such largedoses of hormone to deglycosylate and administer creates yet anotherproblem, namely availability. Large quantities of hormones are presentlyunavailable and could conceivably only be made available throughrecombinant DNA technology.

As mentioned above, deglycosylated hormones, while exhibiting thedesired competitive antagonistic properties in vitro would be of littletherapeutic value in vivo due to their extremely short plasma half-life.The only potential solution to this dilemma would be to preferentiallyremove carbohydrate residues that are responsible for imparting themolecules' biologic action and sparing those that provided themolecules' long plasma half-life. It is, however, impossible to obtainthis result with conventional chemical or enzymatic means (i.e. hydrogenfluoride treatment or enzymatic digestion) because of the non-specificnature of the chemical treatment.

It is an object of the present invention to provide a method forpreferentially removing carbohydrate residues that are responsible forimparting biologic action to molecules while sparing those associatedwith a long plasma half-life.

It is yet another object of the present invention to provide a methodfor obtaining molecules via non-chemical treatment which have thedesired antagonistic nature coupled with a long half-life.

It is still another object of the present invention to provide arecombinant technique for obtaining therapeutically effectiveglycoprotein hormones having been partially deglycosylated by having atleast one oligosaccharide chain entirely removed.

It is still yet another object of the present invention to provide novelhormonal competitive antagonists having therapeutic utility.

It is yet still another object of the present invention to providecompetitive hormone antagonists having substantially batch-to-batchuniformity and consistent potency.

SUMMARY OF THE INVENTION

In accordance with the principles and objects of the present invention,there are provided methods for creating antagonistic molecules byremoving specific glycosylation sites normally present on the moleculethrough a recombinant DNA technique called site-directed mutagenesis.Specifically, the method of the present invention employs the fact thatN-linked oligosaccharides attach to the protein portion of hormones atasparagine residues only when such asparagine residues are present inthe carbohydrate attachment signalling sequence: asparagine--any aminoacid--threonine or serine. The method of the instant inventionadvantageously eliminates the glycosylation site by changing thenucleotide sequence of the gene that codes for the hormone. This isadvantageously accomplished by mutating the codon for asparagineresidues to some other amino acid, preferably one that preserves theoverall charge of the protein or, alternatively, changing the threonineor serine codon to another amino acid thereby removing the glycosylationsignal. The resultant expressed protein hormone will be completelylacking the sugar normally present at the now altered glycosylation sitethereby rendering it a competitive antagonist.

Advantageously, the method allows for the continued specific existenceof other carbohydrate attachment points for prolonging the plasmahalf-life of the molecule without disadvantageously affectingtransduction of the post-receptor bioactivity. Accordingly, and unlikeprior methods, the instant invention can provide a large quantity ofuniform quality, competitive hormone antagonist with a long plasmahalf-life. The invention further comprises therapeutically effectiveformulations of these modified molecules including pharmaceuticallyacceptable salts thereof and also any combination of the foregoing witha carrier vehicle ideally selected to aid in the administration of themolecule to a mammal or other animal without deleteriously affectingeither the molecule or the animal.

Certain specific hormones of the instant invention may also beadvantageously employed as safe and effective first-trimesterabortifacients. After a newly ovulated egg is fertilized within theoviducts, it travels to the uterus where it implants in the innermostlayer, the endometrium. Shortly after implantation, the trophoectodermallayers differentiate into chorionic villi which are finger-likeprojections that "root" deeper within the endometrial layer. As thechorionic villi grow, one cell type within them begins to manufacturelarge amounts of hCG. Until these villi differentiate and proliferateinto a placenta with steroidogenic capacity, the hCG that is secretedtravels to the ovary to "rescue" the corpus luteum from its programmeddemise by maintaining its steroidogenic capacity. Additionally, hCGstimulates the continued secretion of progesterone which preventsuterine contractility and sloughing of the endometrium (menstruation)which would otherwise terminate pregnancy. In contrast, failure tomaintain adequate serum progesterone levels results in spontaneousabortion until after the first trimester of pregnancy when the placentabegins to supply adequate progesterone to maintain the conceptus. Thustreatment of pregnant women, during the first trimester, with an hCGcompetitive antagonist of the present invention will result in a failureof endogenous hCG to maintain the corpus luteum, thereby resulting in afall in serum progesterone levels and abortion.

DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE

Site-directed mutagenesis provides a powerful technique for generatingdefined point mutants. The general procedure, is described by Zoller, M.J. and Smith, M. (1982) Nucl. Acids Res. 10, 6487-6500 and is summarizedas follows: The DNA fragment of interest is cloned into an M13 derivedvector and single-stranded DNA (ssDNA) is prepared. A syntheticoligonucleotide, usually 18±2 bases long and complementary to a regionof the ssDNA except for a mismatch which will direct the mutagenesis, isused as a primer. The template and primer are annealed. Large fragmentE. coli DNA Polymerase I (Klenow), deoxyribonucleotides and T4 DNAligase are added and closed circular double-strandard DNA (ccDNA) issynthesized and ligated. Enrichment for ccDNA is done by one of severalmethods. The enriched ccDNA is transfected with competent JM101 cellsand a population of both mutant and wild-type molecules are obtained.Any of a variety of well known screening procedures may beadvantageously used to differentiate between the mutant and wild-typemolecules. The mutant molecule is then isolated and confirmed bysequencing.

There are several points to consider when employing this method. It ishighly desirable to do a thorough computer search with the proposedoligonucleotide to ensure specific priming at the desired site. Theoligonucleotide should be checked against the M13 vector and the DNAfragment to be mutagenized to be certain this is the case. Anotherpotential difficulty preferably avoided is the increased background ofwild-type molecules resulting from inefficient conversion of ssDNA to dsccDNA. Zoller and Smith reference several methods available to overcomethis potential problem. Zoller and Smith, themselves, employed alkalinesucrose gradients for their enrichment. Similarly, S1 nuclease treatmentmay be used.

In designing the mutagenic oligonucleotide, it is preferable either tocreate or destroy a restriction enzyme site where possible. By so doingthe screening for mutant DNA is greatly facilitated by a simplerestriction site-fragment size analysis.

If the creation or destruction of suitable restriction sites is notpossible, screening may be accomplished by a hybridization assaytechnique using the mutagenic oligonucleotide as a probe. This lattermethod is advantageously independent of the mutation position and can bedone using either phage or isolated DNA. The principle of thehybridization assay is that the mutagenic oligonucleotide will form amore stable duplex with the mutant DNA because they match perfectly;whereas, the wild-type has mismatchina and thus a lower level ofcomplementarity. Advantageously, the hybridization is initially carriedout under conditions of low stringency so that both the mutant andwild-type hybridize to the ³² P-labeled oligonucleotide. The filters arethen washed at increasingly higher temperatures until only the mutantshybridize. This method is very rapid and can be usually carried out inone day. While an isotopic label has been suggested for the probe, otherlabels may be employed although other labels are generally not asdetectable as isotopic labels at very low levels.

EXAMPLE 1 Site-Selection

There are two glycosylation sites in the human α subunit, occurring atamino acid positions 52 and 78. The present invention directs that theamino acid sequence at these positions, Asn-X-Thr be changed toAsn-X-Val, Asn-X-Met or Gln-X-Thr. For "α52", there are several possiblemutations that fit the above scheme all involving 2 base mismatches. Thecodon used for Thr at position 54 is ACC. The codons for Val are GTT,GTC, GTA and GTG; for Met, ATG. So the possibilities are ACC-GTC orACC-ATG. AAC codes for Asn at amino acid 52 and changing this to Gln(CAA or CAG) again involves a 2 base mismatch. Unfortunately, none ofthe above mutations creates a restriction enzyme site. Several sites aredestroyed but these are very common and provide no useful screeninginformation.

For "α78", there are two potential choices for mutation of Thr (ACG). Tochange this codon to Met, ATG, requires a one base substitution. ForVal, GTG, a double mutation is necessary. The Asn-Gln conversionrequires the same changes as in α52. Again, no restriction sites arecreated nor are any destroyed at these positions. However, and as notedpreviously, a 2 base mismatch will be easier to screen for the mutantsince there will be greater homology for its' oligonucleotide than forthe wild-type.

Preferred embodiment for the conversion of the Thr-Met for α78 is givenbelow.

    mutant . . .                                                                                  Asn His Met Ala Cys                                                        AG AAC CAC ATG GCG TGC C                                                   5'                          3'                                      α78    AG AAC CAC ACG GCG TGC C                                                         Asn His Thr Ala Cys                                           oligo          TCTTGGTG TAC CGCACGG                                                     3'                          5'                                  

EXAMPLE 2 Materials

M13mp18 and mp19RF, IPTG, XGal and phenol were obtained from BRL(Bethesda Research Laboratories). T4 DNA ligase, T4 polynucleotidekinase, all restriction endonucleases and E. coli DNA polymerase I largefragment Klenow were purchased from New England Biolabs. ATP wasobtained from Sigma. γ³² P-ATP and α³² P-ATP were purchased fromDuPont/NEN Medical Products. PEG-6000 was obtained from Kodak.Acrylamide, bis-acrylamide and TEMED were purchased from BioRad. S1nuclease, ribonuclease A and E. coli tRNA was obtained from BoehringerMannheim. Deoxyribo-nucleotide triphosphates, pUC 18 and pUC 19 plasmidswere purchased from PL-Pharmacia. Nitrocellulose filters were obtainedfrom Schleicher and Schuell.

Buffers, Media and Stock Solutions

The following buffers were formulated:

    ______________________________________                                        Buffers                                                                       ______________________________________                                        10x kinase   500 mM Tris-Cl (7.5)                                                          100 mM MgCl.sub.2                                                              10 mM DTT                                                                     1 mM spermidine                                                               1 mM EDTA                                                       10x ligase   500 mM Tris-Cl (7.5)                                                          100 mM MgCl.sub.2                                                              10 mM DTT                                                       10x ATP       10 mM ATP, pH 7.0                                               10x dNTP's    2 mM dATP, 2 mM dCTP, 2 mM dGTP,                                              2 mM DTTP                                                                    pH 7.0                                                           Sl "stop"     50 mM Tris base                                                               20 mM EDTA                                                                   lug carrier tRNA                                                 10x Sl       300 mM NaOAC (4.5)                                                            500 mM NaCl                                                                    45 mM ZnSO.sub.4                                                1x TE         10 mM Tris-Cl (8.0)                                                           1 mM EDTA                                                       Solution A   200 mM Tris-Cl (7.5)                                                          100 mM MgCl.sub.2                                                             500 mM NaCl                                                                    10 mM DTT                                                       50x Denhardt's                                                                              1% Ficoll                                                                     1% Polyvinylpyrrilidone                                                       1% BSA                                                          1x SSC       150 mM NaCl                                                                    15 mM Na citrate                                                Media                                                                         5x M9 Salts   10.5 g/L K.sub.2 HPO.sub.4                                                    4.5 g/L KH.sub.2 PO.sub.4                                                     1.0 g/L (NH.sub.4).sub.2 SO.sub.4                                             0.5 g/L Na citrate                                                           Autoclave and temper at 55° C.                                         Add, with sterile technique: 1 ml                                              1M MgSO.sub.4.7 H.sub.2 O; 0.7 ml 0.7%                                       thiamine, 10 ml 20% glucose.                                     ______________________________________                                    

Minimal Media Plates

Add 15 g bactogar to 800 ml ddH₂ O, autoclave. After tempering to 550°C., add 200 ml 5×M9 salts and pour plates.

    ______________________________________                                        YT                                                                            ______________________________________                                        8             g/L bactotryptone                                               5             g/L yeast extract                                               5             g/L NaCl                                                        Autoclave                                                                     ______________________________________                                    

EXAMPLE 3 Preparation of ssDNA

The desired DNA fragment encoding for the alpha subunit was digestedwith the appropriate restriction enzyme and isolated from apolyacrylamide gel. The fragment was ligated into the appropriate sitein the M13 vector of choice via conventional techniques. Approximately3000 ng of fragment and 30 ng digested M13 were mixed in 10× ligasebuffer, 10× ATP and 400U T4 DNA ligase in a total volume of 50 μl. Theligation reaction was incubated for four to six hours at roomtemperature or alternately it can be incubated for 20 hours at 16° C.

A loopful of JM101 cells was transferred from a M9 minimal plate to aflask containing the appropriate volume of YT and grown to a density ofOD₆₆₀ =0.3-0.4 at 37° C. with aeration. The cells were collected bycentrifugation in a Sorvall SS-34 at 8,000 rpm for ten minutes at 4° C.,immediately resuspended in 0.5 volumes of ice-cold 50 mM CaCl₂ and kepton ice for 20 minutes. The cells were centrifuged again and resuspendedin 1/10 volume original growth volume with chilled 50 mM CaCl₂. Thecompetent cells are left on ice until ready to use.

Aliquots of the ligation reaction were added to the JM101competent cellsand transfected as described by Messing, J. (1984) Methods Enzym. 101,20. ssDNA was prepared from individual plaques by growing 5 ml YTcultures with 50 μl exponential JM101 for six to eight hours at 37° C.The cells were centrifuged at 300 rpm for ten minutes at 4° C. and 4 mlsupernatants were transferred to clean Corex tubes. PEG, NaCl and RNaseAwere added to final concentrations of 4%, 500mM and 10 μg/ml,respectively. The tubes were incubated at room temperature for 15minutes. The solutions were then centrifuged at 8,000 rpm for 10 to 15minutes at 4° C. in the SS-34 rotor. The supernatants were decanted andthe pellets resuspended in 300 μl 1×TE and transferred tomicrocentrifuge Eppendorf tubes. A TE-saturated phenol, then etherextractions were done, followed by ethanol precipitation with 1/10volume 3M NaOAC, pH 5.2. The dried pellet was resuspended in TE. Arecombinant wish the desired orientation was then chosen and a one literculture of ssDNA was prepared by scaling-up the above method. This ssDNAserved as template source for all mutagenesis experiments.

EXAMPLE 4-5' Phosphorylation of the Oligonucleotide

For mutagenesis: 1000-3000 pmol of oligonucleotide was phosphorylated ina solution containing 2μl 10× kinase buffer, 2 μl 10× ATP and 4Upolynucleotide kinase in a total volume of 20 μl. The reaction wasincubated at 37° C. for 30-60 minutes and terminated by incubation at65° C. for 10 minutes.

For hydridization: The oligonucleotide was phosphorylated as above in avolume of 50 μl using 50-100uCi γ³² P-ATP as the only source of ATP. Toremove unincorporated nucleotide, the reaction was precipitated with a1/2 volume 7.5M NH₄ OAc and three volumes of cold ethanol. The pelletwas washed several times with cold 70%. ethanol, dried, and resuspendedin 50μl TE.

EXAMPLE 5 Oligonucleotide--Directed Synthesis of ds ccDNA

Annealing: Approximately 13 pmol of ssDNA, 260 pmol 5' phosphorylatedoligonucleotide and 1 μl of solution A were mixed in a total volume of10 μl. As a control, the above was done without the oligonucleotide. Thereactions were incubated at 55° C. for five minutes, then 22° C. forfive minutes.

Extension: 1.5μl 10× Klenow buffer, 1.5 μl 10× dNTP's and 5U Klenow wereadded to the annealing reactions in a final volume of 15μl. Thesereactions were incubated for five minutes at 22° C.

Ligation: To the above reactions, 1 μl 10× ligase, 2 μl 10× ATP and 400UT4 DNA ligase were added to a final volume of 20 μl. The reactions wereincubated for 20 hours at 16° C.

EXAMPLE 6 Enrichment for ccDNA

To the ligation reaction, 3 μl 10× S1 buffer and 0.9U S1 nuclease wereadded to a final volume of 30 μl. A 15 μl aliquot was immediatelyremoved into a 1.5 ml Eppendorf tube containing 40 μl S1 "stop" buffer.The S1 nuclease reaction was allowed to proceed for three minutes atroom temperature. After three minutes, 40μl "stop" buffer was added tothe original reaction eppendorf. The reactions were phenol:CHCI₃extracted and ethanol precipitated. The pellets were then resuspended inTE.

EXAMPLE 7 Transfection of CcDNA

Aliquots of the ligation reactions were used to transfect competentJM101 cells as previously described.

EXAMPLE 8 Screening

A dry nitrocellulose filter was carefully placed on top of eachtransfection plate and placed at 4° C. for 15-30 minutes. The filterswere processed as described by Maniatis, L., Fritsch, E. F. andSambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor. The baked filters were wetted with 2× SSC and prehybridized fortwo or more hours in a solution of 150 mm Tris-Cl(8.0), 750 mM NaCl, 10mM EDTA, 5× Denhardt's, 0.17. SDS and 0.1% sodium pyrophosphate in asealed coaking bag at room temperature. The prehybridization solutionwas removed and replaced with fresh solution containing 1×10⁶ cpm/mlsolution ³² P-labeled oligonucleotide. Hybridization was carried out atroom temperature for 2-20 hours. The filters were then washed with 6XSCC, 0.1% SDS and 0.01 M sodium phosphate (pH 7.2) for 20-30 minutes atincreasing temperatures with autoradiography after each temperaturechange for one hour at -80° C. with two intensifying screens. The finalwashing temperature was based on the equation: T_(m) =2° C.×(the numberof A-T bases)+4° C.×(the number of G-C bases) pursuant to Hanahan, D.and Meselson, M. (1983) Methods Enzym. 100, 333-342. It was observedthat the wild-type was usually off at T_(m) -10° C.

EXAMPLE 9 Isolation of ds Mutant

Single, well-isolated positives were removed into 5 ml YT with 50 μlexponential JM101 as well as streaked into a YT plate with a JM101 lawn.The cultures were incubated at 37° C. with aeration for six to eighthours and then centrifuged at 3,000 rpm for ten minutes at 4° C. ds DNAwas isolated as described in Maniatis, L., Fritsch, E. J. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor. Ifpossible, mutations are confirmed by restriction digests. If not, thescreening procedure is (ideally) repeated on the streaked plates. Alarge culture of the proposed mutant ds DNA was prepared pursuant to themethod described in Maniatis, p.90.

EXAMPLE 10 DNA Sequencing

The double-stranded mutant DNA was then digested with the appropriaterestriction enzymes and isolated from polyacrylamide gels. Thesefragments were end-labeled and sequenced by the method described byMaxam, A. M. and Gilbert, W. (1980) Methods Enzym. 65, 499-560.Alternatively, the fragments can be subcloned into M13 or pUC vectorsand sequenced by the dideoxy method described by Sanger, J., Nicklen, S.and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A 74, 5463-5467.

Transfection of mammalian cells and selection of a cell line produced bythe above procedures can then be expressed by the following procedure:The mutant ds DNA can be ligated with Bam linkers and inserted into abovine papilloma virus (BPV) expression cassette that includes the BPVgenome, the mouse metallothionein promoter and SV40 poly (A) sequencesas per Hsiung et al. (1984) J. Mol. Applied Genetics 2, 497. Mouseepithelioid cells (C127) may then be transfected using the calciumphosphate precipitation method. Transformed cells are ideally identified10-14 days after transfection by their piled-up appearance. Foci ofcells should then be subcloned to obtain a pure population and culturedin Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% fetalbovine serum. Mutant hCG secreted into the culture medium will bepurified by a three step process that included Tris Acryl Bluechromatography, ion exchange and gel filtration chromatography. Activitycan be monitored through the purification process by a radioimmunoassaykit available for this purpose from Serono Laboratories.

The cell line that expresses the greatest amount of mutant hCG willstimulate the secretion of testosterone from suspension cultures ofLeydig cells. Leydig cells can be obtained from adult male mice testes.Approximately 0.25×10⁶ Leydig cells are ideally incubated inapproximately 1 ml (DMEM) that contains one of several dose levels ofnative hCG added to the cells. A half-maximal dose of hCG may then bereadily determined. A second set of suspension cultures should then betreated with a half-maximal dose of hCG and one of several increasingdoses of the mutant recombinant hCG. A biologically inactive form of hCGwill compete for binding to the receptor upon the Leydig cells anddecrease the testosterone response of the native form.

Following the determination of the dose relationship between native andmutant hCG necessary to completely inhibit the native hCG-inducedtestosterone response, the mutant form can then be readily tested forits ability to induce abortion in a variety of species reported tosecrete a chorionic gonadotropin, including the rhesus monkey and theoptional antagonist hormone selected in accordance with the principlesof the present invention.

From the foregoing, one skilled in the art will readily appreciate thatnumerous departures and variations may be made, particularly withrespect to the specific procedures outlined without departing from thespirit or scope of the present invention.

What is claimed is:
 1. A modified glycoprotein hormone which comprises ahuman glycoprotein hormone selected from the group consisting ofluteinizing hormone, follicle stimulating hormone, thyroid stimulatinghormone and chorionic gonadotropin with an N-linked oligosaccharidechain completely eliminated from one but not both of the glycosylationsites on the α-subunit thereof, without removal of an oligosaccharidechain from the β-subunit thereof, and exhibiting approximately the samereceptor binding capacity and plasma half-life as the natural hormone,but sufficiently low post-receptor biological activity to permit its useas a competitive antagonist.
 2. A modified glycoprotein hormoneaccording to claim 1, wherein said modified hormone is a modifiedluteinizing hormone.
 3. A modified glycoprotein hormone according toclaim 1, wherein said modified hormone is a modified folliclestimulating hormone.
 4. A modified glycoprotein hormone according toclaim 1, wherein said modified hormone is a modified thyroid stimulatinghormone.
 5. A modified glycoprotein hormone according to claim 1,wherein said modified hormone is a modified chorionic gonadotropin.
 6. Amodified glycoprotein hormone in accordance with claim 1, wherein saidglycosylation site from which the N-linked oligosaccharide chain hasbeen removed is at the 52 amino acid position.
 7. A human glycoproteinhormone having the amino acid sequence of a human glycoprotein selectedfrom the group consisting of luteinizing hormone, follicle stimulatinghormone, thyroid stimulating hormone and chorionic gonadotropin whereinin place of the Asn-X-Thr residues at one, but not both, of positions52-54 and 78-80 of the alpha-subunit of the natural hormone, there isAsn-X-Val, Asn-X-Met or Gln-X-Thr, wherein said hormone exhibitsapproximately the same receptor binding capacity and plasma half-life asthe natural hormone, but sufficiently low post-receptor biologicalactivity to permit its use as a competitive antagonist.
 8. A hormone inaccordance with claim 7 which differs from the natural hormone in thatthe amino acid residue at position 52 of the alpha-subunit is Gln.
 9. Ahormone in accordance with claim 7 which differs from the naturalhormone in that the amino acid residue at position 78 of thealpha-subunit is Gln.
 10. A hormone in accordance with claim 7 whichdiffers from the natural hormone in that the amino acid residue atposition 54 of the alpha-subunit is Val.
 11. A hormone in accordancewith claim 7 which differs from the natural hormone in that the aminoacid residue at position 54 of the alpha-subunit is Met.
 12. A hormonein accordance with claim 7 which differs from the natural hormone inthat the amino acid residue at position 80 of the alpha-subunit is Val.13. A hormone in accordance with claim 7 which differs from the naturalhormone in that the amino acid residue at position 80 of thealpha-subunit is Met.
 14. A hormone in accordance with claim 7 havingthe beta-subunit of human luteinizing hormone.
 15. A hormone inaccordance with claim 7 having the beta-subunit of human folliclestimulating hormone.
 16. A hormone in accordance with claim 7 having thebeta-subunit of human thyroid stimulating hormone.
 17. A hormone inaccordance with claim 7 having the beta-subunit of human chorionicgonadotropin.